Osteochondral (OC) defects are localized areas of injury or degeneration of articular cartilage and underlying (subchondral) bone resulting in focal and degenerative lesions which if left untreated contribute to irreversible and progressive joint deterioration leading to osteoarthritis (OA). OC defects are challenging to treat as the damage occurs in both articular cartilage and subchondral bone, or more specifically at the OC interface, involving tissues of distinct morphologic and molecular composition. Clinical treatments such as bone marrow stimulation, debridement, autologous chondrocyte implantation, and OC autograft and allograft transplantation may improve clinical symptoms, but do not treat the underlying pathology. Implantable tissue engineered scaffolds that deliver mesenchymal stem cells (MSCs) hold great potential for cell-based OC defect repair as they can be isolated from a variety of adult tissues, are readily expanded in culture, and have been shown to undergo osteogenic and chondrogenic differentiation. Scaffolds embedded with gradients of diffusible growth factors, adhesion ligands, and matrix stiffness have been shown to promote MSC differentiation into chondrogenic and/or osteogenic lineage in 3D culture, however, most of these studies have been limited to stimulating OC differentiation based on gradients of a single factor. We have previously developed novel polymerization approaches that allow for the creation of synthetic hydrogel scaffolds with tunable and continuous gradients of crosslink density and/or elastic modulus, proteolytically mediated degradation, and immobilized cell adhesive peptides (RGD). Furthermore, we have shown that cells respond to these gradients through directed and guided invasion and sprout formation in 3D culture. We hypothesize that spatiotemporal gradients of proteolytic degradation, elastic modulus and integrin-binding peptide ligands can be used to regulate MSC differentiation into osteogenic and chondrogenic lineage. This hypothesis will be addressed by the following specific Aims: Aim 1. To define the effect of spatiotemporal gradients of proteolytic degradation, immobilized RGD concentration, and matrix stiffness on MSC differentiation. Aim 2. To evaluate whether spatiotemporally presented integrin-specific peptide ligands differentially promote MSC differentiation into osteogenic or chondrogenic lineage. These studies will provide the first evidence of how spatiotemporal regulation of multiple types of physiologically relevant gradients modulate MSC integrin expression and lineage commitment in 3D culture in the absence of induction factors. These findings will contribute towards the development of implantable tissue engineered scaffolds that can be used as MSC delivery vehicles to facilitate chondrogenic and osteogenic differentiation for the regeneration of cartilage and bone.